Progranulin (PGRN) is a multifunctional growth factor, also known as PC-cell-derived growth factor (PCDGF), acrogranin, Granulin/epithelin precursor (GEP), proepithelin (PEPI), or GP80, and was first purified as a growth factor from conditioned tissue culture media (Wright W E et al (1989) Cell 56(4):607-617; Zhou J et al (1993) J Biol Chem 268(15):10863-10869). PGRN is a 593-amino-acid secreted glycoprotein with an apparent molecular weight of 88 kDa. PGRN contains seven and a half repeats of a cysteine-rich motif (CX5-6CX5CCX8CCX6CCXDX2HCCPX4CX5-6C) (SEQ ID NO:1) in the order P-G-F-B-A-C-D-E, where A-G are full repeats and P is the half motif (FIG. 24). Notably, PGRN (GEP) undergoes proteolytic processing with the liberation of small, 6-kDa repeat units known as granulins (or epithelins), which retain biological activity (Davidson B et al (2004) Cancer 100(10):2139-2147)). These peptides are active in cell growth assays and may be related to inflammation (Zanocco-Marani, T et al (1999) Cancer Res 59(20):5331-5340; Lu R and Serrero G (2000) Proc Natl Acad Sci USA 97(8):3993-3998).
PGRN has multiple physiological and pathological functions in development, would healing, anti-inflammation, neuron system disorders, as well as cancer. PGRN (GEP) is abundantly expressed in rapidly cycling epithelial cells, in cells of the immune system, and in neurons (Baba T et al (1993) Mol Reprod Dev 34(3):233-243; Daniel R et al (2000) Histochem Cytochem 48(7):999-1009). High levels of GEP expression are also found in several human cancers and contribute to tumorigenesis in diverse cancers, including breast cancer, clear cell renal carcinoma, invasive ovarian carcinoma, glioblastoma, adipocytic teratoma, and multiple myeloma (Davidson B et al (2004) Cancer 100(10):2139-2147; Bateman A et al (1990) Biochem Biophys Res Comm 173(3):1161-1168; Gonzales E M et al (2003) J Biol Chem 278(40):38113-38116; He A and Bateman A (2003) J Mol Med 81(10:600-612; Jones M B et al (2003) Gynecol Oncol 88(1 pt2):S136-139; Wang W et al (2003) Clin Cancer Res 9(6):2221-2228). Although GEP mainly functions as a secreted growth factor, it was also found to be localized inside cells and to directly modulate intracellular activities (Daniel R et al (2000) Histochem Cytochem 48(7):999-1009; Hoque M et al (2003) Mol Cell Biol 23(5):1688-1702). Mutations of PGRN were found to cause frontotemporal lobular degeneration (FTLD) by two groups at the same time (Baker M et al (2006) Nature 442:916-919; Cruts M et al (2006) Nature 442:920-924). Since the initial FTLD studies, 70 pathogenic mutations of PGRN have been reported (reviewed by van Swieten (Van Sweiten J C et al (2008) Lancet Neurol 7(10):965-974) to cause FTLD.
Several PGRN-associated partners have been reported and found to affect PGRN action in various processes. One example is the secretory leukocyte protease inibitor (SLPI). Elastase digests PGRN exclusively in the intergranulin linkers with the generation of granulin peptides. SLPI blocks this proteolysis either by directly binding to elastase or by sequestering granulin peptides from the enzyme (Zhu J et al (2002) Cell 111(6):867-878). PGRN was also found to bind to Sortilin and mediate neurite growth (Hu F et al (2010) Neuron 68:654-667).
Recently, PGRN and PGRN peptides, particularly including the peptide denoted atsttrin, were identified as modulators of TNF/TNFR activity and signaling, and demonstrated to inhibit or block TNF-mediated signaling or response, including TNF-α-induced inflammatory arthritis (Tang W et al (2011) Science 332:478-484; WO 2010120374). Atsttrin is a PGRN-derived engineered protein (Antagonist of TNF/TNFR Signaling via Targeting TNF Receptors), comprising combinations of half units of PGRN units A, C and F in combination with linker units P3, P4 and P5 (U.S. Pat. No. 8,362,218; WO 2010120374). Atsttrin provides a PGRN-derived active peptide having overlapping activity and capability with the full length PGRN molecule. U.S. Pat. No. 8,362,218 and PCT publication WO 2010120374 describe PGRN-derived peptides comprising a combination of half units of progranin/granulin units, wherein at least one half unit is ½ F, and linker units, particularly at least two linker units. The amino acid sequence of PGRN, and PGRN-derived peptides, including attstrin, are depicted in FIGS. 49 and 50.
Lysosomal Storage Diseases
Lysosomes are subcellular organelles responsible for the physiologic turnover of cell constituents. They contain catabolic enzymes, which require a low pH environment in order to function optimally. Lysosomal storage diseases (LSD) describe a heterogeneous group of dozens of rare inherited disorders characterized by the accumulation of undigested or partially digested macromolecules, which ultimately results in cellular dysfunction and clinical abnormalities. LSDs result from gene mutations in one or more of lysosomal enzymes, resulting in accumulation of the enzyme substrates in lysosomes. Organomegaly, connective-tissue and ocular pathology, and central nervous system dysfunction may result. Classically, lysosomal storage diseases encompassed enzyme deficiencies of the lysosomal hydrolases. More recently, the concept of lysosomal storage disease has been expanded to include deficiencies or defects in proteins necessary for the normal post-translational modification of lysosomal enzymes, activator proteins, or proteins important for proper intracellular trafficking between the lysosome and other intracellular compartments.
Over 50 lysosomal storage diseases have been described. The age of onset and clinical manifestations may vary widely among patients with a given lysosomal storage disease, and significant phenotypic heterogeneity between family members carrying identical mutations has been reported. Lysosomal storage diseases are generally classified by the accumulated substrate and include the sphingolipidoses, oligosaccharidoses, mucolipidoses, mucopolysaccharidoses (MPSs), lipoprotein storage disorders, lysosomal transport defects, neuronal ceroid lipofuscinoses and others. FIG. 1 depicts pathways for glycosphingolipids and indicates the altered metabolic enzymes associated with different lysosomal storage diseases.
The most common of the LSDs is Gaucher's Disease, which involves dysfunctional metabolism of sphingolipids and results from hereditary deficiency of the enzyme glucocerebrosidase. Glucocerebrosidase enzyme acts on the fatty acid glucosylceramide and when the enzyme is defective, glucosylceramide accumulates particularly in white blood cells, most often macrophages. Over 300 unique mutations of the glycocerebrosidase encoding gene GBA1 have been identified in Gaucher's Disease (Beutler E and Grabowski G A (2001) Gaucher Disease. in The Metabolic and Molecular Basis of Inherited Disease CR Scriver et al eds. McGraw Hill, N.Y. pp3635-3668; Grabowski G A (2008) Lancet 372(9645):1263-1271; Zhao et al (2003) Clin Genet 64(1):57-64). Glucosylceramide can collect in the spleen, liver, kidneys, lungs, brain and bone marrow.
Gaucher's Disease (GD) falls into three subtypes, with varying pathology and severity. Type I (or non-neuropathic type) is the most common form of the disease, with an incidence of 1 in 50,000 live births of Ashkenazi Jewish heritage. Type I patients have hepatosplenomegaly. The brain is generally not affected pathologically, and depending on disease onset and severity, type 1 patients may live well into adulthood. Many patients have a mild form of the disease or may not show any symptoms. Type I is associated genetically with a GBA1 gene mutation N370S homozygote. Type II (or acute infantile neuropathic Gaucher's disease), begins within 6 months of birth and has an incidence rate of approximately 1 in 100,000 live births. Type II patients have an enlarged liver and spleen, extensive and progressive brain damage, eye movement disorders, spasticity, seizures, limb rigidity, and a poor ability to suck and swallow. Type II patients suffer from serious convulsions, hypertonia, mental retardation and apnea. Affected children usually die by age 2. Type II GD is associated with GBA1 mutation alleles including GBA1 mutation L444P. Type III GD, a chronic neuropathic form, can begin at any time in childhood or even in adulthood, and occurs in approximately 1 in 100,000 live births. It is characterized by slowly progressive but milder neurologic symptoms compared to the acute or type II GD. Major symptoms include an enlarged spleen and/or liver, seizures, poor coordination, skeletal irregularities, eye movement disorders, blood disorders including anemia and respiratory problems. Type III patients suffer from muscle twitches known as myoclonus, convulsions, dementia and ocular muscle apraxia. Patients often live into their early teen years and adulthood. The genetics and any specific GBA1 mutations associated with Type III GD are not clear.
Diagnostic indicators for Gaucher's Disease include increased alkaline phosphatase (ALP), angiotensin-converting enzyme (ACE) and immunoglobulin levels. Alternatively or in addition, cell analysis showing “crinkled paper” cytoplasm and glycolipid-laden macrophages, which are also called “Gaucher's cells” are cellular hallmarks of GD. Mutations in the GBA1 gene are also evaluated, particularly those known to be associated with the disease and Types as noted above. GBA1 mutational analysis can be valuable particularly in families at risk of GD due to family history or that are carriers of GBA1 mutations.
Therapy for LSDs includes enzyme replacement therapy to replace the disease mutant enzyme. Enzyme replacement therapy (ERT) and substrate reduction therapy (SRT) may be applicable for peripheral manifestations in patients with Gaucher disease types I and III, Fabry disease, mucopolysaccharidosis I (Hurler, Hurler-Scheie, and Scheie syndromes), mucopolysaccharidosis II (Hunter syndrome), mucopolysaccharidosis VI (Maroteaux-Lamy syndrome), and Pompe disease. Efforts are underway to develop enzyme replacement options for several other disorders. TABLE 1 provides ERTs being evaluated or approved for treatment of certain LSDs. Exemplary therapies, including ERT, for Gaucher's Disease are listed in TABLE 2. Thus far, ERT has been largely unsuccessful in improving central nervous system manifestations of the lysosomal storage diseases, possibly due to difficulty in penetrating the blood-brain barrier. This has led to active clinical trials evaluating the safety and efficacy of intrathecal enzyme delivery in several lysosomal storage diseases. Also, immune response to enzyme replacement therepay proteins has been reported and can have adverse effects and alter the safety and efficacy of ERT (Brooks D A (1999) Molec Genet Metab 68(2):268-275).
TABLE 1Enzyme Replacement Therapy (ERT) forLysosomal Storage Diseases (LSD)DiseaseEnzyme replacedCompanyStatusGaucher, typeGlucocerebrosidaseGenzymeapproved1 and type 3EU/US(1991)Fabryα-galactosidase AGenzymeapproved EU(2001)approved US(2003)Transkaryoticapproved EUTherapies(2001)MPS I (Hurler)α-L-iduronidaseBioMarinapprovedPharmaceutical/EU/USGenzyme(2003)MPS IVarylsulfatase BBioMarinapproved US(Maroteaux-Pharmaceutical(2005)Lamy)Pompeα-glucosidaseGenzymephase IIIclinical trialMPS IIα-L-iduronateTranskaryoticphase III(Hunter)sulfataseTherapiesclinical trialNiemann-acidGenzymepreclinicalPick BsphingomylinaseMetachromaticarylsulfatase AZymenexpreclinicalleukodystrophyα-Mannosidosis1183α-mannosidaseZymenexpreclinical
TABLE 2Therapies including ERT in Gaucher DiseasesAgentMechanismManufacturerStatusImigluceraseRh GBA1GenzymeFDA approved(ERT)CorporationVelagluceraseRh GBA1Shire plcFDA approvedalfa (ERT)TaliglucerasePlant-derived GBA1Protalix andFDA approvedalpha (ERT)PfizerMiglusta (SRT)InhibitsActelionUnderglucosylceramidedevelopmentsynthaseIsofagomineChaperoning,AmicusUndertartrate (PCT)facilitatesTherapeuticsdevelopmentGBA folding andtrafficking
Therefore, in view of the aforementioned deficiencies attendant with prior art methods of evaluating, ameliorating and treating lysosomal storage diseases, including Gaucher's Disease, it should be apparent that there still exists a need in the art for alternative therapies, additional agents, and improved and more correlative diagnostics for lysosomal storage diseases, including Gaucher's Disease. The present invention provides novel activity, use and application of progranulin (PGRN) and peptide derivatives thereof including atsttrin, including in diagnosis, amelioration, and treatment of lysosomal storage diseases, including Gaucher's Disease.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.